Small molecule inhibitors of ghrelin O-acyltransferase

ABSTRACT

Ghrelin O-acyltransferase (GOAT) is inhibited with designed small molecules of the general formula: 
                         
Methods comprise contacting the GOAT with an inhibitor and detecting a resultant inhibition.

This application is a continuation of Ser No. 12/571,538, filed Oct. 1,2009 (now, U.S. Pat. No. 8,013,015), which claims priority to Ser. No.61/102,353, filed Oct. 2, 2008.

This work was supported by grants from the National Institutes of Health(HL20948); the Government has certain rights in this invention.

The field of the invention is inhibition of ghrelin O-acyltransferase(GOAT).

INTRODUCTION

Ghrelin O-acyltransferase (GOAT) is the membrane-bound enzyme thatattaches 8-carbon octanoate to a serine residue in ghrelin, a peptidehormone. Ghrelin comprises the N-terminal 28 amino acids of proghrelin,a precursor of 94 amino acids that is cleaved proteolytically to releaseghrelin. The octanoylated serine is the third amino acid from theN-terminus of ghrelin, and its presence is essential for the physiologicactions of ghrelin in stimulating appetite and other neuroendocrinefunctions.

Most ghrelin is produced in a minor population of endocrine cells in thegastric mucosa, originally called X/A-like cells and now called ghrelincells. After secretion into plasma, octanoylated ghrelin travels to thepituitary where it binds to a G protein-coupled receptor that triggersthe release of growth hormone. The plasma concentration of ghrelin risesimmediately before meals, and the octanoylated peptide enhances foodintake when administered to rats and humans. Inactivation of the geneencoding ghrelin or its receptor produces a modest resistance to obesityin mice that are presented with a high fat diet. These observations ledto the hypothesis that interference with ghrelin action might protectagainst obesity in humans.

One way to inhibit ghrelin action would be to inhibit GOAT. The enzymeis an attractive target because ghrelin is the only protein known to beoctanoylated, and hence GOAT inhibition is likely to alter only oneprotein. GOAT was first identified by transfection of candidate cDNAsinto cultured endocrine cells that process proghrelin to ghrelin butfail to attach octanoyl. With these transfection assays, the enzymeattached octanoyl to the appropriate serine-3 in ghrelin, and theattachment reaction was shown to require the two amino acids in GOAT(asparagine-307 and histidines-338) that are conserved in othermembrane-bound O-acyltransferases. GOAT is a highly hydrophobic proteinwith eight postulated membrane-spanning helices. It is presumed to belocated in the endoplasmic reticulum where proghrelin is initiallyinserted.

Thus far, the action of GOAT has been studied only in intact cells. Incopending U.S. Ser No. 12/167,917, we described a biochemical assay forGOAT activity using membranes from insect cells that express mammalianGOAT as a result of infection with a recombinant baculovirus. Usingpurified recombinant proghrelin as a substrate, we characterized theenzymatic properties of GOAT, and we showed that the enzyme is potentlyinhibited by octanoylated peptides that derived from the first fiveamino acids of ghrelin. Here we disclose the design and synthesis ofsmall molecule GOAT inhibitors.

SUMMARY OF THE INVENTION

The invention provides methods, compounds and compositions forinhibiting ghrelin O-acyltransferase (GOAT). In one embodiment, theinvention provides compounds of and pharmaceutical compositionscomprising a GOAT inhibitor having the structure 1:

wherein:

R₁, is selected from optionally hetero-, optionally substituted (C6-C9)alkyl, optionally hetero-, optionally substituted (C6-C9) alkenyl, andoptionally hetero-, optionally substituted (C6-C9) alkynyl;

R₂-R₅ are independently selected from an electron pair, hydrogen,optionally hetero, optionally substituted alkyl, optionally hetero-,optionally substituted alkenyl, optionally hetero-, optionallysubstituted alkynyl, optionally hetero-, optionally substituted aryl,and an optionally substituted heteroatom;

A is selected from CH₂, O, S, NH, and N-alkyl;

G is selected from O, S, NH, and N-alkyl;

X is selected from hydroxyl, amino, alkylamino, and alkylthio;

Y is selected from C, N, O and S (wherein the X—Y bond encompasses bothdiastereomers); and

Z is selected from CH₂, O, S, NH and N-alkyl.

The invention encompasses all alternative combinations of particularembodiments:

-   -   wherein R₁, is selected from optionally hetero-, optionally        substituted hepta-alkyl, optionally hetero-, optionally        substituted hepta-alkenyl, and optionally hetero-, optionally        substituted hepta-alkynyl;    -   wherein R₁, is selected from hepta-alkyl (heptanyl),        hepta-alkenyl (heptenyl), and hepta-alkynyl (heptynyl);    -   wherein R₁, is selected from n-heptanyl, n-heptenyl, and        n-heptynyl;    -   wherein R₁, is n-heptanyl;    -   wherein R₁, is n-4-heptenyl (—CH₂CH₂CH₂CHCHCH₂CH₃) (E or Z        isoform);    -   wherein R₁, is n-4-heptynyl;    -   wherein R₁ is heteroalkyl, comprising an oxygen heteroatom;    -   wherein R₁ is methyl-diethylene glycol (—CH2OCH2CH2OCH2CH3);    -   wherein R₂ is H or optionally substituted, lower (C1-C5) alkyl;    -   wherein R₂ is methyl, ethyl, propyl or butyl;    -   wherein R₃ is H or optionally substituted, lower (C1-C5) alkyl;    -   wherein R₃ is H, methyl, ethyl, propyl or butyl;    -   wherein R₄ is methoxy;    -   wherein R₄ has the structure 2:

wherein R₇ is selected from a bond, optionally substituted lower alkyl,NH, S and O; R₈ and R₉ are independently selected from hydrogen andoptionally hetero-, optionally substituted alkyl; and D is selected fromC, N, and R₁₀ is a 5-7 membered, optionally heterocyclic ring,particularly wherein R₈ is H and R₉ is methylmethoxy (—CH₂OCH₃);

-   -   wherein R₅ is R₆(CH₂)n wherein R₆ is a 5-7 membered, optionally        heterocyclic ring, and n is an integer from 0 to 5;    -   wherein R₅ is benzyl;    -   wherein A is CH₂, O, S, NH or N—(C1-C3) alkyl;    -   wherein G is O, S, NH or N—(C1-C3) alkyl;    -   wherein X is hydroxyl, amino, (C1-C4) alkylamino, or (C1-C4)        alkylthio;    -   wherein Y is C, N, O or S (wherein the X—Y bond encompasses both        diastereomers);    -   wherein Z is CH₂, O, S, NH or N—(C1-C3) alkyl;    -   wherein the inhibitor is selected from structures set forth        herein, particularly BK1114, BK1165, PH1152 and the compounds of        Table 1.

The subject compositions may be co-formulated or coadministered orcoprescribed with an additional active ingredient such as (i) anappetite-suppressant or antiobesity drug such as Orlistat, Sibutramine,Metformin, Byetta, Symlin, and Rimonabant; (ii) an anti-diabetic drugsuch as (a) Insulin; (b) Secretagogues including Sulfonylureas such asglipizide (Glucotrol), glyburide (Diabeta, Micronase, Glynase),glimepiride (Amaryl), gliclazide (Diamicron) and Meglitinides, such asrepaglinide (Prandin) and nateglinide (Starlix) (c) Sensitizersincluding Biguanides such as metformin and Thiazolidinediones such asrosiglitazone and pioglitazone; (d) Alpha-glucosidase inhibitors such asmiglitol (Glyset) and acarbose (Precose/Glucobay); and (e) Peptideanalogs including Incretin mimetics, Glucagon-like peptide (GLP) analogsand agonists such as Exenatide and Liraglutide, Gastric inhibitorypeptide (GIP) analogs, DPP-4 inhibitors such as vildagliptin andsitagliptin, and Amylin analogues; or (iii) a cholesterol-modulatingdrug such as HMG-CoA reductase inhibitors.

The invention also encompasses methods of formulating, using the subjectcompounds and compositions, including methods of inhibiting GOAT,comprising the step of contacting the GOAT with a subject compound orcomposition, and optionally, detecting a resultant inhibition of theGOAT. The target GOAT is usually endogenous, in vivo, in a patientdetermined to be in need of GOAT inhibition, such as patients determinedor diagnosed to be suffering from obesity, diabetes,hypercholesterolemia, etc.

DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

The following descriptions are provided by way of illustration only andnot by way of limitation. Those skilled in the art will readilyrecognize a variety of noncritical parameters that could be changed ormodified to yield essentially similar results.

The following descriptions of particular embodiments and examples areoffered by way of illustration and not by way of limitation. Unlesscontraindicated or noted otherwise, in these descriptions and throughoutthis specification, the terms “a” and “an” mean one or more, the term“or” means and/or and polynucleotide sequences are understood toencompass opposite strands as well as alternative backbones describedherein. Furthermore, genuses are recited as shorthand for a recitationof all members of the genus; for example, the recitation of (C1-C3)alkyl is shorthand for a recitation of all C1-C3 alkyls: methyl, ethyland propyl, including isomers thereof.

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N), sulfur (S) and silicon (Si).

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which is fully saturated,having the number of carbon atoms designated (i.e. C1-C8 means one toeight carbons). Examples of alkyl groups include methyl, ethyl,n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl and the like.

The term “alkenyl”, by itself or as part of another substituent, means astraight or branched chain, or cyclic hydrocarbon radical, orcombination thereof, which may be mono- or polyunsaturated, having thenumber of carbon atoms designated (i.e. C2-C8 means two to eightcarbons) and one or more double bonds. Examples of alkenyl groupsinclude vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl) and higher homologs and isomersthereof.

The term “alkynyl”, by itself or as part of another substituent, means astraight or branched chain hydrocarbon radical, or combination thereof,which may be mono- or polyunsaturated, having the number of carbon atomsdesignated (i.e. C2-C8 means two to eight carbons) and one or moretriple bonds. Examples of alkynyl groups include ethynyl, 1- and3-propynyl, 3-butynyl and higher homologs and isomers thereof.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from alkyl, as exemplified by—CH2-CH2-CH2-CH2-. Typically, an alkyl (or alkylene) group will havefrom 1 to 24 carbon atoms, with those groups having 10 or fewer carbonatoms being preferred in the invention. A “lower alkyl” or “loweralkylene” is a shorter chain alkyl or alkylene group, generally havingeight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and from one to three heteroatoms selectedfrom the group consisting of O, N, Si and S, wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S may be placed atany interior position of the heteroalkyl group. The heteroatom Si may beplaced at any position of the heteroalkyl group, including the positionat which the alkyl group is attached to the remainder of the molecule.Examples include —CH2CH2-O—CH3, —CH2-CH2-NH—CH3, —CH2-CH2-N(CH3)-CH3,—CH2-S—CH2-CH3, —CH2-CH2, —S(O)—CH3, —CH2-CH2-S(O)2-CH3, —CH═CH—O—CH3,—Si(CH3)₃, —CH2-CH═N—OCH3, and —CH═CH—N(CH3)-CH3. Up to two heteroatomsmay be consecutive, such as, for example, —CH2-NH—OCH3 and—CH2-O—Si(CH3)3.

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent means a divalent radical derived from heteroalkyl, asexemplified by —CH2-CH2-S—CH2-CH2- and —CH2-S—CH2-CH2-NH—CH2-. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Accordingly, acycloalkyl group has the number of carbon atoms designated (i.e., C3-C8means three to eight carbons) and may also have one or two double bonds.A heterocycloalkyl group consists of the number of carbon atomsdesignated and from one to three heteroatoms selected from the groupconsisting of O, N, Si and S, and wherein the nitrogen and sulfur atomsmay optionally be oxidized and the nitrogen heteroatom may optionally bequaternized. Additionally, for heterocycloalkyl, a heteroatom can occupythe position at which the heterocycle is attached to the remainder ofthe molecule. Examples of cycloalkyl include cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include 1-(1,2,5,6-tetrahydropyrid-yl), 1-piperidinyl,2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl,tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl,tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” and “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include alkyl substituted with halogen atoms, which can be thesame or different, in a number ranging from one to (2m′+1), where m′ isthe total number of carbon atoms in the alkyl group. For example, theterm “halo(C1-C4)alkyl” is mean to include trifluoromethyl,2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like. Thus,the term “haloalkyl” includes monohaloalkyl (alkyl substituted with onehalogen atom) and polyhaloalkyl (alkyl substituted with halogen atoms ina number ranging from two to (2m′+1) halogen atoms, where m′ is thetotal number of carbon atoms in the alkyl group). The term“perhaloalkyl” means, unless otherwise stated, alkyl substituted with(2m′+1) halogen atoms, where m′ is the total number of carbon atoms inthe alkyl group. For example the term “perhalo(C1-C4)alkyl” is meant toinclude trifluoromethyl, pentachloroethyl,1,1,1-trifluoro-2-bromo-2-chloroethyl and the like.

The term “acyl” refers to those groups derived from an organic acid byremoval of the hydroxy portion of the acid. Accordingly, acyl is meantto include, for example, acetyl, propionyl, butyryl, decanoyl, pivaloyl,benzoyl and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,typically aromatic, hydrocarbon substituent which can be a single ringor multiple rings (up to three rings) which are fused together or linkedcovalently. Non-limiting examples of aryl groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl and 1,2,3,4-tetrahydronaphthalene.

The term heteroaryl,” refers to aryl groups (or rings) that contain fromzero to four heteroatoms selected from N, O, and S, wherein the nitrogenand sulfur atoms are optionally oxidized and the nitrogen heteroatom areoptionally quaternized. A heteroaryl group can be attached to theremainder of the molecule through a heteroatom. Non-limiting examples ofheteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyland 6-quinolyl.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) is meant to include both substituted and unsubstitutedforms of the indicated radical. Preferred substituents for each type ofradical are provided below.

Substituents for the alkyl and heteroalkyl radicals (as well as thosegroups referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl,alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl andheterocycloalkenyl) can be a variety of groups selected from: —OR′, ═O,═NR′, ═N—OR′, —NR′R″, —SW, halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′,—CO2R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″,—NR′—SO2NR′″, —NR″CO2R′, —NH—C(NH2)=NH, —NR′C(NH2)=NH, —NH—C(NH2)=NR′,—S(O)R′, —SO2R′, —SO2NR′R″, —NR″SO2R, —CN and —NO2, in a number rangingfrom zero to three, with those groups having zero, one or twosubstituents being particularly preferred. R′, R″ and R″ eachindependently refer to hydrogen, unsubstituted (C1-C8)alkyl andheteroalkyl, unsubstituted aryl, aryl substituted with one to threehalogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, oraryl-(C1-C4)alkyl groups. When R′ and R″ are attached to the samenitrogen atom, they can be combined with the nitrogen atom to form a 5-,6- or 7-membered ring. For example, —NR′R″ is meant to include1-pyrrolidinyl and 4-morpholinyl. Typically, an alkyl or heteroalkylgroup will have from zero to three substituents, with those groupshaving two or fewer substituents being preferred in the invention. Morepreferably, an alkyl or heteroalkyl radical will be unsubstituted ormonosubstituted. Most preferably, an alkyl or heteroalkyl radical willbe unsubstituted. From the above discussion of substituents, one ofskill in the art will understand that the term “alkyl” is meant toinclude groups such as trihaloalkyl (e.g., —CF3 and —CH2CF3).

Preferred substituents for the alkyl and heteroalkyl radicals areselected from: —OR′, ═O, —NR′R″, —SR′, halogen, —SiR′R″R′″, —OC(O)R′,—C(O)R′, —CO2R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR″CO2R′,—NR′—SO2NR″R′″, —S(O)R′, —SO2R′, —SO2NR′R″, —NR″SO2R, —CN and —NO2,where R′ and R″ are as defined above. Further preferred substituents areselected from: —OR′, ═O, —NR′R″, halogen, —OC(O)R′, —CO2R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR″CO2R′, —NR′—SO2NR″R′″, —SO2R′, —SO2NR′R″,—NR″SO2R, —CN and —NO2.

Similarly, substituents for the aryl and heteroaryl groups are variedand selected from: halogen, —OR′, —OC(O)R′, —NR′R″, —SW, —R′, —CN, —NO2,—CO2R′, —CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —NR″CO2R′,—NR′—C(O)NR″R′″, —NR′—SO2NR″R′″, —NH—C(NH2)=NH, —NR′C(NH2)=NH,—NH—C(NH2)=NR′, —S(O)R′, —SO2R′, —SO2NR′R″, —NR″SO2R, —N3, —CH(Ph)2,perfluoro(C1-C4)alko-xy and perfluoro(C1-C4)alkyl, in a number rangingfrom zero to the total number of open valences on the aromatic ringsystem; and where R′, R″ and R′″ are independently selected fromhydrogen, (C1-C8)alkyl and heteroalkyl, unsubstituted aryl andheteroaryl, (unsubstituted aryl)-(C1-C4)alkyl and (unsubstitutedaryl)oxy-(C1-C4)alkyl. When the aryl group is1,2,3,4-tetrahydronaphthalene, it may be substituted with a substitutedor unsubstituted (C3-C7)spirocycloalkyl group. The(C3-C7)spirocycloalkyl group may be substituted in the same manner asdefined herein for “cycloalkyl”. Typically, an aryl or heteroaryl groupwill have from zero to three substituents, with those groups having twoor fewer substituents being preferred in the invention. In oneembodiment of the invention, an aryl or heteroaryl group will beunsubstituted or monosubstituted. In another embodiment, an aryl orheteroaryl group will be unsubstituted.

Preferred substituents for aryl and heteroaryl groups are selected from:halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN, —NO2, —CO2R′, —CONR′R″,—C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —S(O)R′, —SO2R′, —SO2NR′R″, —NR″SO2R,—N3, —CH(Ph)2, perfluoro(C1-C4)alkoxy and perfluoro(C1-C4)alkyl, whereR′ and R″ are as defined above. Further preferred substituents areselected from: halogen, —OR′, —OC(O)R′, —NR′R″, —R′, —CN, —NO2, —CO2R′,—CONR′R″, —NR″C(O)R′, —SO2R′, —SO2NR′R″, —NR″SO2R,perfluoro(C1-C4)alkoxy and perfluoro(C1-C4)alkyl.

The substituent —CO2H, as used herein, includes bioisostericreplacements therefor; see, e.g., The Practice of Medicinal Chemistry;Wermuth, C. G., Ed.; Academic Press: New York, 1996; p. 203.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CH2)q-U-, wherein T and U are independently —NH—, —O—, —CH2- ora single bond, and q is an integer of from 0 to 2. Alternatively, two ofthe substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula -A-(CH2)r-B—,wherein A and B are independently —CH2-, —O—, —NH—, —S—, —S(O)—,—S(O)2-, —S(O)2NR′— or a single bond, and r is an integer of from 1 to3. One of the single bonds of the new ring so formed may optionally bereplaced with a double bond. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula —(CH2)s-X—(CH2)t-, where s and t areindependently integers of from 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—,—S(O)2-, or —S(O)2NR′—. The substituent R′ in —NR′— and —S(O)2NR′— isselected from hydrogen or unsubstituted (C1-C6)alkyl.

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds which are prepared with relatively nontoxicacids or bases, depending on the particular substituents found on thecompounds described herein. When compounds of the invention containrelatively acidic functionalities, base addition salts can be obtainedby contacting the neutral form of such compounds with a sufficientamount of the desired base, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable base addition salts includesodium, potassium, calcium, ammonium, organic amino, or magnesium salt,or a similar salt. When compounds of the invention contain relativelybasic functionalities, acid addition salts can be obtained by contactingthe neutral form of such compounds with a sufficient amount of thedesired acid, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable acid addition salts include those derivedfrom inorganic acids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived fromrelatively nontoxic organic acids like acetic, propionic, isobutyric,oxalic, maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic,phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric,methanesulfonic, and the like. Also included are salts of amino acidssuch as arginate and the like, and salts of organic acids likeglucuronic or galactunoric acids and the like (see, for example, Bergeet al. (1977) J. Pharm. Sci. 66:1-19). Certain specific compounds of theinvention contain both basic and acidic functionalities that allow thecompounds to be converted into either base or acid addition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the invention.

In addition to salt forms, the invention provides compounds which are ina prodrug form. Prodrugs of the compounds described herein are thosecompounds that undergo chemical changes under physiological conditionsto provide the compounds of the invention. Additionally, prodrugs can beconverted to the compounds of the invention by chemical or biochemicalmethods in an ex vivo environment. For example, prodrugs can be slowlyconverted to the compounds of the invention when placed in a transdermalpatch reservoir with a suitable enzyme or chemical reagent. Prodrugs areoften useful because, in some situations, they may be easier toadminister than the parent drug. They may, for instance, be morebioavailable by oral administration than the parent drug. The prodrugmay also have improved solubility in pharmacological compositions overthe parent drug. A wide variety of prodrug derivatives are known in theart, such as those that rely on hydrolytic cleavage or oxidativeactivation of the prodrug. An example, without limitation, of a prodrugwould be a compound of the invention which is administered as an ester(the “prodrug”), but then is metabolically hydrolyzed to the carboxylicacid, the active entity. Additional examples include peptidylderivatives of a compound of the invention.

Certain compounds of the invention can exist in unsolvated forms as wellas solvated forms, including hydrated forms. In general, the solvatedforms are equivalent to unsolvated forms and are intended to beencompassed within the scope of the invention. Certain compounds of theinvention may exist in multiple crystalline or amorphous forms. Ingeneral, all physical forms are equivalent for the uses contemplated bythe invention and are intended to be within the scope of the invention.

Certain compounds of the invention possess asymmetric carbon atoms(optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are all intended to beencompassed within the scope of the invention.

The compounds of the invention may also contain unnatural proportions ofatomic isotopes at one or more of the atoms that constitute suchcompounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds ofthe invention, whether radioactive or not, are intended to beencompassed within the scope of the invention.

The term “therapeutically effective amount” refers to the amount of thesubject compound that will elicit, to some significant extent, thebiological or medical response of a tissue, system, animal or human thatis being sought by the researcher, veterinarian, medical doctor or otherclinician, such as when administered, is sufficient to preventdevelopment of, or alleviate to some extent, one or more of the symptomsof the condition or disorder being treated. The therapeuticallyeffective amount will vary depending on the compound, the disease andits severity and the age, weight, etc., of the mammal to be treated.

The invention also provides pharmaceutical compositions comprising thesubject compounds and a pharmaceutically acceptable excipient,particularly such compositions comprising a unit dosage of the subjectcompounds, particularly such compositions copackaged with instructionsdescribing use of the composition to treat a disease associated withundesirably high GOAT activity, particularly as found in obesity.

Accordingly, the invention provides methods of treating a diseaseassociated with undesirable GOAT activity, the method comprising thestep of administering an effective dosage of the subject compounds andcompositions, which may be followed by the step of detecting a resultantdecrease in pathology associated with the disease, and which may beprefaced by the step of diagnosis such disease and/or prescribing suchcomposition. Applicable disease include obesity, diabetes andhypercholesterolemia.

The invention also provides methods of inhibiting a GOAT, the methodcomprising the step of contacting a composition comprising a GOAT withan effective amount of the subject compounds and compositions, which maybe followed by the step of detecting a resultant change in GOATactivity.

The compositions for administration can take the form of bulk liquidsolutions or suspensions, or bulk powders. More commonly, however, thecompositions are presented in unit dosage forms to facilitate accuratedosing. The term “unit dosage forms” refers to physically discrete unitssuitable as unitary dosages for human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect, in association with asuitable pharmaceutical excipient. Typical unit dosage forms includeprefilled, premeasured ampules or syringes of the liquid compositions orpills, tablets, capsules, losenges or the like in the case of solidcompositions. In such compositions, the inhibitor is usually a minorcomponent (from about 0.1 to about 50% by weight or preferably fromabout 1 to about 40% by weight) with the remainder being variousvehicles or carriers and processing aids helpful for forming the desireddosing form.

Suitable excipients or carriers and methods for preparing administrablecompositions are known or apparent to those skilled in the art and aredescribed in more detail in such publications as Remington'sPharmaceutical Science, Mack Publishing Co, NJ (1991). In addition, theinhibitors may be advantageously used in conjunction with othertherapuetic agents as described herein or otherwise known in the art.Hence the compositions may be administered separately, jointly, orcombined in a single dosage unit.

The amount administered depends on the inhibitor formulation, route ofadministration, etc. and is generally empirically determined in routinetrials, and variations will necessarily occur depending on the target,the host, and the route of administration, etc. Generally, the quantityof active compound in a unit dose of preparation may be varied oradjusted from about 1, 3, 10 or 30 to about 30, 100, 300 or 1000 mg,according to the particular application. In a particular embodiment,unit dosage forms are packaged in a multipack adapted for sequentialuse, such as blisterpack, comprising sheets of at least 6, 9 or 12 unitdosage forms. The actual dosage employed may be varied depending uponthe requirements of the patient and the severity of the condition beingtreated. Determination of the proper dosage for a particular situationis within the skill of the art. Generally, treatment is initiated withsmaller dosages which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small amounts until the optimumeffect under the circumstances is reached. For convenience, the totaldaily dosage may be divided and administered in portions during the dayif desired.

The following are examples (Formulations 1-4) of inhibitor capsuleformulations.

TABLE 1 Capsule Formulations Capsule Formula 1 Formula 2 Formula 3Formula 4 Formulation mg/capsule mg/capsule mg/capsule mg/capsuleInhibitor (Solid 100 400 400 200 Solution) Silicon Dioxide 0.625 2.53.75 1.875 Magnesium 0.125 0.5 0.125 0.625 Stearate NF2 Croscarmellose11.000 44.0 40.0 20.0 Sodium NF Pluronic F68 6.250 25.0 50.0 25.0 NFSilicon Dioxide 0.625 2.5 3.75 1.875 NF Magnesium 0.125 0.5 1.25 0.625Stearate NF Total 118.750 475.00 475.00 475.00 Capsule Size No. 4 No. 0No. 0 No. 2

Preparation of Solid Solution

Crystalline inhibitor (80 g/batch) and the povidone (NF K29/32 at 160g/batch) are dissolved in methylene chloride (5000 mL). The solution isdried using a suitable solvent spray dryer and the residue reduced tofine particles by grinding. The powder is then passed through a 30 meshscreen and confirmed to be amorphous by x-ray analysis.

The solid solution, silicon dioxide and magnesium stearate are mixed ina suitable mixer for 10 minutes. The mixture is compacted using asuitable roller compactor and milled using a suitable mill fitted with30 mesh screen. Croscarmellose sodium, Pluronic F68 and silicon dioxideare added to the milled mixture and mixed further for 10 minutes. Apremix is made with magnesium stearate and equal portions of themixture. The premix is added to the remainder of the mixture, mixed for5 minutes and the mixture encapsulated in hard shell gelatin capsuleshells.

The inhibitors can be administered by a variety of methods including,but not limited to, parenteral, topical, oral, or local administration,such as by aerosol or transdermally, for prophylactic and/or therapeutictreatment. Also, in accordance with the knowledge of the skilledclinician, the therapeutic protocols (e.g., dosage amounts and times ofadministration) can be varied in view of the observed effects of theadministered therapeutic agents on the patient, and in view of theobserved responses of the disease to the administered therapeuticagents.

The therapeutics of the invention can be administered in atherapeutically effective dosage and amount, in the process of atherapeutically effective protocol for treatment of the patient. Formore potent inhibitors, microgram (ug) amounts per kilogram of patientmay be sufficient, for example, in the range of about 1, 10 or 100 ug/kgto about 0.01, 0.1, 1, 10, or 100 mg/kg of patient weight though optimaldosages are compound specific, and generally empirically determined foreach compound.

In general, routine experimentation in clinical trials will determinespecific ranges for optimal therapeutic effect, for each therapeutic,each administrative protocol, and administration to specific patientswill also be adjusted to within effective and safe ranges depending onthe patient condition and responsiveness to initial administrations.However, the ultimate administration protocol will be regulatedaccording to the judgment of the attending clinician considering suchfactors as age, condition and size of the patient as well as inhibitorpotency, severity of the disease being treated. For example, a dosageregimen of the inhibitors can be oral administration of from 10 mg to2000 mg/day, preferably 10 to 1000 mg/day, more preferably 50 to 600mg/day, in two to four (preferably two) divided doses. Intermittanttherapy (e.g., one week out of three weeks or three out of four weeks)may also be used.

In Vitro Octanoylation Assay Details.

The assay we used is described in copending U.S. Ser No. 12/167,917,filed: Jul. 3, 2008. For baculoviral infection of insect cells, mouseGOAT cDNA was inserted into pFastBac HT-A (His10-tag) (Invitrogen), andbaculovirus was generated. Sf9 insect cells were cultured, set up on day0 at a density of 5×10⁵ cells/ml, and infected on day 1 with baculovirusencoding His 10-GOAT at a density of 1×10⁶ cells/ml. Cells wereharvested 48 h post-infection and washed once with phosphate-bufferedsaline.

Each pellet of Sf9 cells (obtained from 1 liter of cell culture) wasdisrupted with a Dounce homogenizer (40 strokes) on ice in 50 ml ofbuffer containing 50 mM Tris-Cl (pH 7.0), 150 mM NaCl, 1 mM sodium EDTA,1 mM dithiothreitol, 100 μM bis(4-nitrophenyl) phosphate, 2.5 μg/mlaprotinin, 20 μg/ml phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin,and 10 μg/ml pepstatin A. After an initial centrifugation at 3,000 g for5 min at 4° C., the supernatant was further centrifuged at 100,000 g for30 min at 4° C. to obtain a membrane fraction, which was stored at −80°C. until the time of assay. Just prior to assay, the membranes weresuspended in 50 mM HEPES-NaOH (pH 7.0) and then passed through a22-gauge needle 10 times. After centrifugation at 1,000 g for 1 min toremove aggregated materials, the supernatant (hereafter referred asmembranes) was used for assays.

Unless otherwise stated, the standard assay mixture contained 50 mMHEPES-NaOH (pH 7.0), 50 μM palmitoyl CoA or myristyl ether CoA, 50 μgmembrane protein, 5 μg recombinant wild type or mutant versions ofproghrelin-His8 and 1 μM [³H]octanoyl CoA (11 dpm/fmol) in a finalvolume of 50 μl. After incubation at 37° C. for 5 min, each reaction wasstopped by the addition of 1 ml of buffer A (50 mM Tris-Cl at pH 7.5,150 mM NaCl, and 0.1% (w/v) SDS), after which each sample was loadedonto a 0.2-ml nickel-affinity column (QIAGEN). The columns were washedat room temperature with 1 ml of buffer A followed by 3 ml of buffer Acontaining 25 mM imidazole. The His-tagged proghrelin was then elutedwith 1 ml of buffer A containing 250 mM imidazole. Radioactivity presentin the 250 mM-imidazole eluate was quantified by liquid scintillationcounting.

Design and Synthesis of Small Molecule GOAT Inhibitors

While details of the GOAT catalyzed acylation are not yet clear,biochemical data is consistent with an intermediate of type 1A beinggenerated at some point along the reaction coordinate. In thismechanism, the serine-3 hydroxyl group of GOAT bound ghrelin would bedeprotonated by an active site base (e.g. histidine). The resultantalkoxide would add to the carbonyl carbon of either an acylated enzymeresidue or to the thioester of separately bound octanoyl CoA. Species 1Athus formed would presumably resemble the transition state for thereaction. Enzymes are thought to enhance reaction rates (forward andback) by lowering the energy required to reach the transition state (TS)of the processes they catalyze. This is achieved, in part, by the TSgeometry being bound favorably. Tight binding but non-reactive mimics ofTS geometries spawned the field of catalytic antibodies as originallyenvisioned by Pauling, and they have also become useful as specificenzyme inhibitors.

In the GOAT forward reaction, we reasoned that 1A would collapse torelease R- and ester product. The enzyme is selective for transferringan eight-carbon fatty acid, and conserved ghrelin residues flankingserine-3 are required for efficient catalysis. We combined theseelements into a single design. Namely, we would synthesize compoundsable to bind GOAT in a manner similar to the tetrahedral oxyanion in1A—and where the tetrahedral geometry at that site would be stable (1B).The molecules would further possess a hydrophobic segment of appropriatesize to exploit GOAT selectivity for octanoylation. Lastly, themolecules would contain sufficient additional functionality to occupypart of the (pro)ghrelin binding space—wherein favorable interactionssuch as hydrogen-bonding could further stabilize binding and slow thedissociation rate of an enzyme/inhibitor complex.

Replacing O4 in 1A with carbon while choosing R3 (1B) to be a poorleaving group was considered a means to access transition state mimeticsfor the acyl transfer. We targeted an (S)-2-amino-5-hydroxy lauric acidcore (1B) that could be elaborated as needed. There was no literaturedescribing syntheses of such compounds and therefore a de novopreparation was developed.

We transformed commercial L-methionine methyl ester hydrochloride into(S)—N—Z-vinylglycine (2) using Rapoport's protocols (Scheme 1) andsubsequently cross metathesized this material with racemic 1-decene-3-ol(3) using Grubbs' ruthenium alkylidene A as catalyst. The resultantcomposite product (PH1147) was then hydrogenolyzed in MeOH to affordamino alcohol PH1148. This material was condensed independently with3-hydroxy-3-methylbutyric acid and 2,2-dimethyl-3-hydroxy propionic acidto give PH1149 and PH1150, respectively. The latter was saponified inaqueous LiOH and the derived acid condensed with commercial(S)-(+)-α-(methoxymethyl)phenethylamine (B) to afford PH1152.

This completed our initial set of potential GOAT inhibitors. Eachcompound was isolated as an ˜1:1 mixture of C5 epimers (the result ofusing racemic decenol in the metathesis with optically active 2). Theepimers were not separated because, if the intended mode of inhibitionwere to operate, wherein the inhibitor's secondary alcohol would mimicthe tetrahedral oxyanion in 1, the GOAT active site would presumablyaccommodate one C5 diastereomer better than the other, yet there was noway to predict which one at this stage

Compounds PH1147-1152 were screened for GOAT inhibition in vitro usingmembrane fractions prepared from Sf9 insect cells infected withbaculovirus encoding mouse GOAT. The assay (described in U.S. Ser. No.12/167,917) measures the extent of specific acyl transfer from tritiatedoctanoyl CoA to recombinant His-tagged proghrelin. PH1147, PH1149,PH1150 and PH1152 inhibited GOAT activity at a high concentration (500μM) with PH1147 and PH1152 showing the greatest inhibition, and whentitrated to lower concentrations, PH1147 performed best (IC₅₀˜90 μM).PH1147 was the simplest molecule in the set and lacked motifs present inthe more complex analogs intended to be Ser2 and/or Phe4 mimetics.

To probe the series further, we hydrogenated PH1147 in the presence ofcatalytic amounts of Crabtree's iridium complex C (Scheme 2). Unlike thehydrogenolysis of PH1148 wherein both the olefin and thebenzyloxycarbonyl groups were reduced, this alcohol-directed reductionusing C selectively saturates the alkene to afford PH1154. Competingisomerization of the alkene to an enol in situ was also observed andPH1154 was produced alongside ketone PH1154a. Fortunately the two werereadily separable by silica gel chromatography. With PH1154 in hand, thematerial was saponified with aqueous LiOH and the incipient acidcondensed with (S)-(+)-α-(methoxymethyl)phenethylamine (B) to affordPH1156. Both PH1154a and PH1156 inhibit GOAT comparably to PH1147 at 100μM concentrations in vitro; however, they performed more poorly athigher concentrations.

It appeared perhaps the benzoxylcarbonyl (a.k.a. Z) protecting group inPH1147 was actually a beneficial feature. We noted also that PH1147 wasthe one compound in the series retaining the trans alkene from themetathesis reaction. To explore these issues, we removed the Z groupfrom PH1156 to afford PH1159 and reduced the ester of PH1147 to providediol PH1165. The latter was elaborated to PH1167 wherein anN-methylglycine-alanine dipeptide was installed as a potential surrogatefor gly1 and ser2 of ghrelin. However, neither PH1159 nor PH1167possessed activity comparable to PH1147 while PH1165 was only slightlyless active. These observations supported the idea that the Z groupcontributed positively to inhibition. Adding segments of ghrelin peptidein place of the Z group was not helpful nor was replacing it with atrifluoroacetyl unit (see BK1112). Still, PH1147 activity was weak andwe needed an indication that inhibition was consistent with known GOATpreferences.

Along these lines we synthesized BK1114 by cross metathesizing vinylglycine 2 with racemic 3-hydroxy-3-methyl-1-decene (4), itself preparedby homologating 2-nonanone with dimethylsulfonium methylide (Scheme 3).BK1114 is identical to PH1147 except that the C5 hydrogen atom isreplaced with a methyl group. This substitution allowed us to beginprobing tolerance in the C5 region, which corresponds to ‘R’ in 1 andwould be either the sulfur of CoA

or a non-hydrogen atom from a GOAT active site residue. BK1114 isroughly twice as efficacious as PH1147 in inhibiting GOAT in vitro at100 μm concentration. Furthermore, beginning with 2 and3-hydroxy-methyl-1-pentene (5) we synthesized BK1095. This molecule isidentical to BK1114 except its hydrocarbon chain is truncated by fiveatoms. BK1095 shows little inhibition of GOAT activity at allconcentrations tested—suggesting the system is responding to hydrocarbonchain length consistent with GOAT selectivity for octanoylation.

During this time it was discovered that GOAT activity was subject toend-product inhibition and that replacing the ester linkage inoctanoylated ghrelin(1-28) with the corresponding amide improvedperformance significantly. This same trend held with octanylated ghrelinpentapeptides. The first five N-terminal residues having serine-3replaced by (S)-2,3-diaminopropionic acid and the distal amine of thisunit octanoylated (Structure 2) was an inhibitor showing an IC₅₀ of ˜1.5μM in vitro. It was essential to cap the C-terminus as a primarycarboxamide in this case. We decided to examine similar C-terminalextensions in our series.

We saponified BK1114 with aqueous LiOH and condensed the resultant acidwith commercial Phe-Leu-CONH₂ hydrochloride to afford BK1129 (Scheme 4).It was possible to separate the C5 diastereomers of this material bypreparative HPLC. The two isomers were assayed independently for GOATinhibition. Neither was as effective as BK1114 in terms of potency. Wehypothesized that the Z group in BK1114 was binding to GOAT such thatits phenyl group occupied a binding pocket reserved for Phe4 of ghrelin(Scheme 5). Such binding would presumably require rotation about Cα-Cβbond in BK1114 wherein S stereochemistry at Cα would actually appearunnatural to the enzyme. Moreover, it would orient functionality slatedto occupy ghrelin Phe5 and Leu5 space in the opposite direction. Wetested this idea by synthesizing BK1149. This molecule is derived fromD-methionine and is epimeric at Cα relative to BK1114. We alsosynthesized BK1135 wherein the methyl ester was removed entirely.

BK1149 retains activity in vitro comparable to BK1114 while BK1135 isless potent. We also synthesized BK1137 and BK1141 wherein the Z groupwas replaced with the β-hydroxy-α,α′-dimethylpropionamide unit firstused in PH1152 (see Scheme 1).

BK1141 has a phenylalanine unit and retains some activity howeverBK1137, which lacks both the Z group and a phenylalanine residue, isessentially inactive. Taken together, the data our postulated bindingmode for BK1114 and suggest in such an orientation the stereochemistryat Cα is not specifically recognized by the enzyme (i.e. BK1114 andBK1149 are similarly efficacious).

We have used an alcohol group as a mimic of the tetrahedral oxyanion inthe putative transition state for acyl transfer. Alternative functionalgroups can potentially fill this role; for example, the knownhomocysteine dimer 6 was reductively alkylated with iodooctane in thepresence of tributylphosphine (Scheme 5). The resultantS-octylhomocysteine derivative was oxidized to the correspondingsulfoxide BK1145 with sodium periodate. Dialkyl sulfoxides maintain atetrahedral geometry and are chiral at sulfur. BK1145 was isolated as amixture of diastereomers. While not as potent as BK1114, the moleculedoes inhibit GOAT in a dose responsive manner.

We also examined octanoylated (S)-2,3-diaminopropionic ester LN1111622as a simplified version of DapOctanoylghrelin(1-5)CONH₂. This moleculewould presumably not function as a transition state inhibitor but ratherbe a surrogate for the product of GOAT activity. The compound does notperform as well as BK1114.

In summary, ispropylamide analogs of BK1114 (namely PH1176a & PH1176b)do not improve activity; however, the C5 methyl group can be replacedwith propyl without compromising activity (see BK1165).

Inhibition of GOAT Activity by BK1114 in Intact Cells.

A stably transfected INS-1 cell line co-expressing preproghrelin andGOAT was established as described below. On day 0, cells were set up ata density of 1.5×10⁶/100-mm dish in medium A (RPMI-1640 mediumsupplemented with 10 mM HEPES, 50 μM β-mercaptoethanol, 100 U/mlpenicillin, and 100 μg/ml streptomycin) containing 10% (v/v) FCS. On day2, fresh medium was added. On day 4, the cells received a directaddition of the indicated concentration of BK1114 delivered in DMSO. Thefinal concentration of DMSO in each dish was 0.5%. 24 h after treatment,two dishes of cells from each condition were harvested and pooled, afterwhich peptides were extracted from the cells, fractioned onreverse-phase chromatography, and subjected to immunoblot analysis witha 1:1000 dilution of rabbit anti-ghrelin antibody as previouslydescribed (Yang J, Brown M S, Liang G, Grishin N V, Goldstein J L (2008)Identification of the acyltransferase that octanoylates ghrelin, anappetite-stimulating peptide hormone. Cell 132:387-39). Films wereexposed for 1 min.

Stably transfected cell line co-expressing preproghrelin and GOAT. RatINS-1 cells were grown in monolayer at 37° C. in 8.8% CO₂ as previouslydescribed (Id.). On day 0, cells were plated at a density of5×10⁵/100-mm dish in medium A (see above) containing 10% (v/v) FCS. Onday 1, cells were transfected with 5 μg of pCMV-preproghrelin usingFuGENE HD Transfection Reagent (Roche) according to the manufacture'sinstructions. 24 h after transfection, the cells were switched to mediumA containing 10% FCS and 400 μg/ml G418 to select the cells expressingthe neo-containing pCMV-preproghrelin plasmid. Fresh medium was addedevery 2 to 3 days until colonies formed at ˜14 days. Individual colonieswere isolated with cloning cylinders and then subcloned by dilutionplating. Expression of proghrelin and ghrelin was assessed by immunoblotanalysis. After the establishment of the cell line expressingpreproghrelin, the preproghrelin-expressing cells were set up on day 0at a density of 5×10⁵/100-mm dish in medium A containing 10% (v/v) FCS.On day 1, cells were transfected with 3 μg of pEF-GOAT-T7 using FuGENEHD Transfection Reagent (Roche) according to the manufacture'sinstructions. 24 h after transfection, the cells were switched to mediumA containing 10% FCS and 0.2 μg/ml puromycin to select the cellsexpressing the pac-containing pEF-GOAT-T7 plasmid. Subcloning of thecell line was the same as mentioned above. Expression of GOAT-T7 wasassessed by immunoblot analysis. The results of these experimentsdemonstrated a BK1114 dose inhibition response from 5 to 10 to 20 to 50uM inhibitor.

Inhibition of GOAT activity by [Dap³]octanoyl-ghrelin (1-5)-NH2 peptideand BK1114. Membranes from Sf9 cells infected with baculovirus encodingmouse His₁₀-GOAT were prepared as previously described (Yang J, Zhao TJ, Goldstein J L, Brown M S. 2008 PNAS, 105: 10750-10755). Each 50-μlreaction mixture contained 50 μg of membrane protein, 5 μg ofproghrelin-His₈, 50 μM palmitoyl CoA, 1 μM http[³H]octanoyl CoA (11dpm/fmol), and the experimental concentrations of peptide or compound ina final concentration of 3% (vol/vol) DMSO. Control values wereestablished which represent the amount of [3H]octanoyl proghrelin formedin the absence of compound. The results of demonstrated dose responseinhibition by both the pentapeptide and by BK1114.

Inhibition of GOAT activity by the synthetic compounds, BK1114, BK1176,BK1177 and BK1183. Membranes from Sf9 cells infected with baculovirusencoding mouse His₁₀-GOAT were prepared as previously described (Yang etal., 2008, supra). Each 50-μl reaction mixture contained 50 μg ofmembrane protein, 5 μg of proghrelin-His₈, 50 μM palmitoyl CoA, 1 μM[³H]octanoyl CoA (11 dpm/fmol), and the experimental concentrations ofthe compound in a final concentration of 3% (vol/vol) DMSO. Controlvalues were established which represent the amount of [3H]octanoylproghrelin formed in the absence of compound. The results demonstrateddose response inhibition by each of the test compounds.

Organic Synthesis

BK1114. Trimethylsulfonium iodide (23.53 g, 115.3 mmol) was suspended inanhydrous THF (250 mL) under argon and the slurry cooled to −20° C. Apre-cooled (−20° C.) solution of n-BuLi (2.14M in hexanes, 51.2 mL, 109mmol) was added slowly via cannulating needle and the solution stirredfor 10 minutes at −20° C. A pre-cooled (−20° C.) solution 2-nonanone(5.0 mL, 28.8 mmol) was then added slowly via cannulating needle and theresultant solution was stirred at −20° C. for 1 hour. The reaction wasallowed to warm to room temperature over 2 hours. Water (200 mL) wasadded and the mixture was extracted with Et₂O (3×100 mL). The combinedorganics were dried over MgSO4, filtered and concentrated in vacuo toafford an oily residue. This material was chromatographed on silica gel(eluting with 10% EtOAc/hexanes) to afford 3-hydroxy-3-methyl-1-decene(3.56 g, 72% yield) as a colorless oil.

N—Z—(S)-vinylglycine (2) was prepared from L-methionine as describedpreviously [Rapoport et al. J. Org. Chem. (1980) 45, 4817].

N—Z—(S)-vinylglycine (2: 55 mg, 0.22 mmol) and3-hydroxy-3-methyl-1-decene (74 mg, 0.44 mmol) were each dissolved in0.3 mL anhydrous 1,2-dichloroethane. The two solutions were addedseparately, and simultaneously, dropwise by syringe to a solution ofruthenium complex A (Aldrich Chemical Co., 13.7 mg, 0.021 mmol) in 0.5mL 1,2-dichlorethane under an argon atmosphere. When the additions werecomplete, the resultant dark solution was warmed to reflux and stirredat that temperature for 18 hours. The solution was cooled to ambienttemperature, concentrated in vacuo and the residue obtained waschromatographed on silica gel (eluting with 25% EtOAc/hexanes) to affordBK1114 as a colorless oil (˜1:1 mixture of C5 diastereomers, 26 mg, 30%yield).

Variations of the aforementioned procedures are available to provideanalogs. For example, replacing 3-hydroxy-3-methyl-1-decene with allylicalcohol 6 in the cross-metathesis with 2 will provide a product whosealkyne protecting group can be removed by oxidative decomplexation toafford 7. Further variations of 7 wherein the alkyne unit occupies otherpositions within the hydrocarbon chain are similarly accessible.

Variations of the aforementioned procedures are available to provideanalogs. For example, replacing 3-hydroxy-3-methyl-1-decene with allylicalcohol 8 in the cross-metathesis with 2 will provide product 9.

Variations of the aforementioned procedures are used to provide furtheranalogs. For example, cross-methathesizing heterocycle 10 withfluorinated allylic alcohol 11 will provide product 12.

Variations of the aforementioned procedures are use to provide furtheranalogs. For example, replacing 3-hydroxy-3-methyl-1-decene with allylicamine 13 in the cross-metathesis with 2 will provide product 14.

BK1165 can be manipulated to produce additional analogs. For example,hydrogenolysis in the presence of catalytic amounts of palladium oncarbon, treating the incipient amine with carbamoyl chloride 15 followedby dissolution in isopropanol in the presence of a transesterificationcatalyst will afford 16. Myriad variations of such procedures areavailable to generate related structures.

TABLE 1 Structures of additional, exemplary active analogs.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A pharmaceutical composition comprising a ghrelin O-acyltransferase(GOAT) inhibitor of structure 1:

wherein: R₁ is selected from n-heptanyl, n-heptenyl, n-heptynyl, andmethyl-diethylene glycol (—CH₂OCH₂CH₂OCH₂CH₃); R₃-R₅ are independentlyselected from an electron pair, hydrogen, optionally hetero-, optionallysubstituted alkyl, optionally hetero-, optionally substituted alkenyl,optionally hetero-, optionally substituted alkynyl, optionally hetero-,optionally substituted aryl, and an optionally substituted heteroatom; Ais selected from CH₂, O, S, NH, and N-alkyl; G is selected from O, S,NH, and N-alkyl; X is selected from hydroxyl, amino, alkylamino, andalkylthio; Y is C; and Z is selected from CH₂, O, S, NH and N-alkyl, ora pharmaceutically acceptable salt thereof, wherein said inhibitorinhibits said ghrelin O-acyltransferase (GOAT), and said composition issuitable for pharmaceutical use, wherein R₂ is optionally-substituted,lower (C1-C5) alkyl.
 2. The composition of claim 1 wherein R₁ isn-heptanyl.
 3. The composition of claim 1 wherein R₂ is methyl, ethyl,propyl or butyl.
 4. The composition of claim 1 wherein R₃ is H oroptionally substituted, lower (C1-C5) alkyl.
 5. The composition of claim1 wherein R₃ is H, methyl, ethyl, propyl or butyl.
 6. The composition ofclaim 1 wherein R₄ is methoxy.
 7. The composition of claim 1 wherein R₄is of structure 2:

wherein: R₇ is the point of attachment and is selected from a bond,optionally substituted lower alkyl, NH, S and O; R₈ and R₉ areindependently selected from hydrogen and optionally hetero-, optionallysubstituted alkyl; and D is selected from C, N, and R₁₀ is a 5-7membered, optionally heterocyclic ring.
 8. The composition of claim 1wherein R₅ is R₆(CH₂)_(n) wherein R₆ is a 5-7 membered, optionallyheterocyclic ring, and n is an integer from 0 to
 5. 9. A pharmaceuticalcomposition comprising a ghrelin O-acyltransferase (GOAT) inhibitor ofstructure 1:

wherein: R₁ is selected from n-heptanyl, n-heptenyl, n-heptynyl, andmethyl-diethylene glycol (—CH₂OCH₂CH₂OCH₂CH₃); R₂, R₃ and R₅ areindependently selected from an electron pair, hydrogen, optionallyhetero-, optionally substituted alkyl, optionally hetero-, optionallysubstituted alkenyl, optionally hetero-, optionally substituted alkynyl,optionally hetero-, optionally substituted aryl, and an optionallysubstituted heteroatom; A is selected from CH₂, O, S, NH, and N-alkyl; Gis selected from O, S, NH, and N-alkyl; X is selected from hydroxyl,amino, alkylamino, and alkylthio; Y is C; and Z is selected from CH₂, O,S, NH and N-alkyl, or a pharmaceutically acceptable salt thereof,wherein said inhibitor inhibits said ghrelin O-acyltransferase (GOAT),and said composition is suitable for pharmaceutical use, wherein R₄ ismethoxy.
 10. The composition of claim 9 wherein R₁ is n-heptanyl. 11.The composition of claim 9 wherein R₂ is optionally-substituted, lower(C1-C5) alkyl.
 12. The composition of claim 9 wherein R₂ is methyl,ethyl, propyl or butyl.
 13. The composition of claim 9 wherein R₃ is Hor optionally substituted, lower (C1-C5) alkyl.
 14. The composition ofclaim 9 wherein R₃ is H, methyl, ethyl, propyl or butyl.
 15. Thecomposition of claim 9 wherein R₅ is R₆(CH₂)_(n) wherein R₆ is a 5-7membered, optionally heterocyclic ring, and n is an integer from 0 to 5.16. A pharmaceutical composition comprising a ghrelin O-acyltransferase(GOAT) inhibitor of structure 1:

wherein: R₁ is selected from n-heptanyl, n-heptenyl, n-heptynyl, andmethyl-diethylene glycol (—CH₂OCH₂CH₂OCH₂CH₃); R₂-R₄ are independentlyselected from an electron pair, hydrogen, optionally hetero-, optionallysubstituted alkyl, optionally hetero-, optionally substituted alkenyl,optionally hetero-, optionally substituted alkynyl, optionally hetero-,optionally substituted aryl, and an optionally substituted heteroatom; Ais selected from CH₂, O, S, NH, and N-alkyl; G is selected from O, S,NH, and N-alkyl; X is selected from hydroxyl, amino, alkylamino, andalkylthio; Y is C; and Z is selected from CH₂, O, S, NH and N-alkyl, ora pharmaceutically acceptable salt thereof, wherein said inhibitorinhibits said ghrelin O-acyltransferase (GOAT), and said composition issuitable for pharmaceutical use, wherein R₅ is R₆(CH₂)_(n) wherein R₆ isa 5-7 membered, optionally heterocyclic ring, and n is an integer from 0to
 5. 17. The composition of claim 16 wherein R₁ is n-heptanyl.
 18. Thecomposition of claim 16 wherein R₂ is optionally-substituted, lower(C1-C5) alkyl.
 19. The composition of claim 16 wherein R₂ is methyl,ethyl, propyl or butyl.
 20. The composition of claim 16 wherein R₃ is Hor optionally substituted, lower (C1-C5) alkyl.
 21. The composition ofclaim 16 wherein R₃ is H, methyl, ethyl, propyl or butyl.
 22. Thecomposition of claim 16 wherein R₄ is methoxy.
 23. The composition ofclaim 16 wherein R₄ is of structure 2:

wherein: R₇ is the point of attachment and is selected from a bond,optionally substituted lower alkyl, NH, S and O; R₈ and R₉ areindependently selected from hydrogen and optionally hetero-, optionallysubstituted alkyl; and D is selected from C, N, and R₁₀ is a 5-7membered, optionally heterocyclic ring.
 24. A pharmaceutical compositioncomprising a ghrelin O-acyltransferase (GOAT) inhibitor or apharmaceutically acceptable salt thereof, wherein said inhibitor is of astructure selected from the group consisting of:

PH1147

PH1149

PH1150

PH1154

PH1154a

PH1156

PH1159

PH1165

PH1167

BK1112

BK1129

BK1149

BK1135

BK1141

BK1145

LN1111622

PH1176a

PH1176b


25. A method of inhibiting ghrelin O-acyltransferase (GOAT), comprisingthe steps of contacting the GOAT with the composition of claim 9, anddetecting a resultant inhibition of the GOAT.
 26. A method of inhibitingghrelin O-acyltransferase (GOAT), comprising the steps of contacting theGOAT with the composition of claim 9, and detecting a resultantinhibition of the GOAT.
 27. A method of inhibiting ghrelinO-acyltransferase (GOAT), comprising the steps of contacting the GOATwith the composition of claim 16, and detecting a resultant inhibitionof the GOAT.
 28. A method of inhibiting ghrelin O-acyltransferase(GOAT), comprising the steps of contacting the GOAT with the compositionof claim 24, and detecting a resultant inhibition of the GOAT.